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Effects of gonadotropin-releasing hormone agonist on brain development and aging: results from two animal models
Syed M. Nuruddin
Thesis for the Degree of Doctor of Philosophy (PhD)
Faculty of Veterinary Medicine and Biosciences Department of Production Animal Clinical Sciences
Oslo, 2013
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CONTENTS
Acknowledgment ... 4
Summary ... 6
Sammendrag (Norwegian) ... 9
Abbreviations ... 12
List of papers ... 15
Introduction ... 16
Gonadotropin- releasing hormone and its receptor ... 17
Functional roles of mammalian GnRH and its receptors ... 18
Functions in the reproductive system ... 18
Function in the central nervous system ... 20
Functional roles of hippocampus and amygdala in cognition ... 22
Hippocampus ... 22
Amygdala ... 23
Role of GnRH and its receptor in cognitive function ... 25
GnRH agonist ... 26
Application of GnRH agonist in adults and their side effects ... 26
Usage of GnRHa in pediatric medicine ... 27
Aim of the thesis ... 29
Materials and Methods ... 31
Animals and treatment ... 31
Methods ... 33
Behaviour study - Spatial orientation task ... 34
Hippocampal gene expression involved in synaptic plasticity and endocrine signaling ... 35
Postmortem magnetic resonance image (MRI)) for morphometric analysis of global and regional brain volumes ... 36
Transcription profiling through microarray in amygdala samples ... 38
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mRNA Expression of GnRHI and GnRHR in the hippocampus of Transgenic mice ... 40
Measurement of amyloid-β deposition in cerebral cortex, thalamus and hippocampus using immunohistochemistry ... 40
Statistical analyses ... 42
Results; summery of the papers ... 46
Paper I ... 46
Paper II ... 47
Paper III ... 48
Paper IV ... 49
Discussion ... 50
Methodological considerat ions ... 51
Animal models ... 51
Spatial orientation test ... 54
Microarray ... 55
Quantitative real time pcr (qRT-PCR) ... 57
Magnetic resonance image (MRI) ... 57
Immunocytohistochemistry ... 59
Experimental findings ... 60
GnRHa treatment impact in spatial orientation as well as hippocampal genes involved in synaptic plasticity and endocrine signaling ... 60
GnRHa treatment effect on amygdalar volume of the brain ... 63
Impact of GnRHa treatment on gene expression changes in the amygdala ... 66
GnRHa treatment effect on gene expression changes and amyloid plaques deposition ... 69
Relevance of findings for human medicine ... 70
Conclusions ... 71
Further studies ... 72
References ... 74 Paper I-IV
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Acknowledgment
I want to take this opportunity to thank my colleagues, family and friends for their support over the last few years throughout my PhD, and also to thank the Norwegian School of Veterinary Science, Norwegian Research Council, by Health Authorities South East of Norway (2007), AstraZeneca London (2008) and Welcome Trust for funding my PhD project. Firstly I would like to thank Associate Professor Anette Krogenæs, Professor Erik Ropstad, Professor Ira Haraldsen, Professor Neil Evans, Professor Jane Robinson and Professor Mahmood Amiry- Moghaddam for all of their supervision, guidance and encouragement throughout my PhD. It has been a pleasure working together for the past four years. Particularly, I am grateful to Ira and Erik for introducing me to the world of scientific research. Ira and Erik, your ambition and passion for science have been truly inspiring. Your guidance throughout the writing process has proven invaluable and has contributed substantially to the quality of my work. Most of all, I would like to thank my all supervisors for your generosity and hospitality and for inviting me to come and work with you in this wonderful and beautiful country. Erik; I really appreciated your warm welcome at the Oslo central bus station, when I arrived in Oslo for the first time.
Anette you were not only my supervisor, you took care of me in every aspects from scientific writing to family issues as well as inspiring me to never ever give up in science.
Neil and Jane; Thanks for your welcome and giving me the opportunity to work in your lab at the University of Glasgow, Scotland.
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Mahmood; you were truly inspiring person for me and thanks for your all encouragement during my PhD.
I would also like to thank other members of the SOBER (Sex on Brain European Research), group; Swavek Wojniusz andMuriel Bruchhage, for their help and skills needed to carry out my PhD.
Many thanks also go to my other colleagues Nina Hårdnes, Almås Camilla, Melanie Koenig for help with microarray, quantitative real time PCR (qRT-PCR) experiments.
Finally I would like to thank my family and friends for their kind and valuable support throughout the past four years. In particular I would like to thank my wife Hasina for her support and patience listening to the daily failures and triumphs for my PhD.
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Summary
Normal brain maturation is the result of many structural and molecular changes that can be modulated by endocrine variables and is associated with brain plasticity and sex- and age specific differences in cognitive performance. In many species, sexual dimorphisms in brain structure and function have been documented, some of which are present at birth but some of which develop post-natally. Using an ovine model, the work contained in this thesis demonstrates that a peri-pubertal pharmacological blockade of gonadotropin-releasing hormone (GnRH) action (by chronic treatment with a gonadotropin releasing hormone agonist (GnRHa)) results in increased sex-differences in emotional behavior and other cognitive functions. One of the aims of this body of work was to determine what changes in brain structure and function were present in such GnRHa treated animals. The hippocampus is the most investigated brain region with regard to the complex interaction of memory and spatial orientation, functions which are thought to be sexually differentiated.
The aim of the study described in paper I was, therefore, to investigate whether peri-pubertal GnRHa treatment had an effect on a hippocampus dependent cognitive task, namely spatial orientation and whether the pharmacological blockade of GnRH affected expression of hippocampal genes associated with endocrine signaling and synaptic plasticity. The GnRHa treatment had no significant effect on spatial orientation ability although there was a tendency of females to perform better than males; however, GnRHa treatment was associated with significant sex- and hemisphere specific changes in mRNA expression for some of the investigated genes.
The aim of the study described in Paper II, was to investigate the effect of GnRHa treatment on structural development of the ovine brain such as total brain, hippocampus and amygdala volume
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using magnetic resonance image (MRI). Analysis revealed highly significant GnRHa treatment effects on the volume of the left and right amygdalae, indicating larger amygdalae in treated animals. Significant sex differences were found for total grey matter and the right amygdala, indicating larger volumes in male compared to female animals. Additionally, we observed a significant interaction between sex and treatment on left amygdala volume, indicating stronger effects of treatment in female compared to male animals.
The aim of the study described in Paper III, was to investigate the molecular mechanisms that underlie these GnRHa-induced morphological changes in the amygdala using Agilent ovine microarray technologies to identify genes affected by GnRHa treatment followed by qRT-PCR to verify the noted changes in gene expression. Gene network analysis was performed to predict the functional impact of the differentially expressed genes. The analysis demonstrated that GnRHa treatment was associated with significant sex- and hemisphere specific differential expression of genes in treated female, but not in treated male animals.
Recent studies have indicated an association between hormones of the hypothalamic–pituitary–
gonadal (HPG) axis and cognitive senescence, suggesting that post meno-/andropausal changes in HPG hormones are involved in cognitive and neuropathological changes associated with aging such as in Alzheimer´s Disease (AD). GnRH and luteinizing hormone (LH) have long been shown to have central roles in reproductive physiology, however, GnRH receptors are also highly expressed in brain regions that are affected in AD (temporal-hippocampal cortex and limbic system), and thus age related changes in GnRH signaling could have an impact on amyloid deposits in AD. The aim of the study described in paper IV was therefore to investigate the effect of intervention with a GnRHa on amyloid plaque deposition and GnRH and GnRHR
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mRNA expression in a double transgenic mouse model that is predisposed to AD due to the presence of Arctic and Swedish amyloid beta (A4) precursor protein mutations (tg-ArcSwe).
Analysis showed that the GnRHa/transgenes treatment clearly affects gene expression both at hormone and receptor level, while the effect GnRHa on amyloid plaque development remained unclear.
Overall, these findings substantiate the need for further studies investigating neurobiological effects of GnRH and the potential neurobiological side effects of GnRHa treatment on the brain in animals and humans.
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Sammendrag (Norwegian)
Normal utvikling av hjernen er et resultat av mange strukturelle og molekylære endringer som påvirkes av endokrine variabler og er assosiert med hjernens plastisitet og kjønns – og alders spesifikke forskjeller i kognitive prestasjoner. Seksuell dimorphisme i hjernens struktur og funksjon er dokumentert hos mange arter, noe er til stede ved fødselen mens noe utvikles postnatalt. Ved å bruke en ovin modell, har arbeidet i denne avhandlingen vist at en peri-pubertal farmakologisk blokkering av gonadotropin frisettende hormon (GnRH) med en GnRH agonist (GnRHa), resulterte i økte kjønnsforskjeller i emosjonell adferd og andre kognitive funksjoner.
Et av målene var å bestemme forandringer i hjernens struktur og funksjon hos GnRHa behandlete dyr. Hippocampus er det mest undersøkte hjerneavsnittet med tanke på den komplekse interaksjonen som fins mellom hukommelse og orientering i rom (spatial orientation), funksjoner sett på som seksuelt differensierte.
Målet med studiet i artikkel 1 var derfor å undersøke om peri-pubertal GnRHa behandling hadde en effekt på en typisk hippocampus avhengig kognitiv oppgave som orientering i rom, og om den farmakologiske blokkeringen av GnRH påvirket uttrykk av gener i hippocampus assosiert med endokrin signalisering og synaptisk plastisitet. GnRHa behandling hadde ingen signifikant effekt på evnen til orientering i rommet, selv om det var en tendens til at hunndyr var bedre enn hanndyr, men GnRHa behandling var assosiert med signifikante kjønns – og hemisfære spesifikke forandringer i mRNA uttrykk for noen av de undersøkte genene.
Målet med studiet i artikkel 2, var å undersøke effekten av GnRHa behandling på den strukturelle utviklingen av den ovine hjernen. Volum av hele hjernen, hippocampus og amygdala
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ble undersøkt ved hjelp av magnetic resonance image (MRI). Det var signifikant økt volum av venstre og høyre amygdala hos behandlete dyr. Det ble funnet signifikante kjønnsforskjeller for total grå substans og høyre amygdala med større volum hos hanndyr enn hunndyr. I tillegg observerte vi en signifikant interaksjon mellom kjønn og behandling når det gjaldt volum av venstre amygdala, med større effekt av behandling hos hunndyr enn hanndyr.
Målet med studiet i artikkel 3 var å undersøke de molekylære mekanismene som ligger bak disse GnRHa- induserte morfologiske forandringene i amygdala. Agilent ovine microarray teknologier ble brukt for å identifisere gener som ble affisert av GnRHa behandling, etterfulgt av qRT-PCR for å verifisere endringene i genekspresjonen. Gen nettverk analyser ble utført for å forutsi mulig funksjonell betydning av genene med endret uttrykk. Analysen viste at GnRHa behandling var assosiert med en signifikant kjønns- og hemisfære spesifikk differensiert ekspresjon av gener i behandlete hunndyr, men ikke i behandlete hanndyr.
Nyere studier har indikert en assosiasjon mellom hormoner i hypothalamus-hypofyse-gonade (HPG) akse og kognitive prestasjoner ved begynnende aldring, hvilket indikerer at forandringer etter meno-/andropausen i HPG hormoner er involvert i kognitive og nevropatologiske forandringer assosiert med aldring som f.eks. i Alzheimers sykdom (AD). Det har vært kjent lenge at GnRH og luteiniserende hormon (LH) har sentrale roller i reproduksjonsfysiologien.
Imidlertid er GnRH reseptorer også sterkt uttrykt i hjerneavsnitt som affiseres i AD (temporal- hippocampal cortex og limbiske system), og alders relaterte forandringer i GnRH signalisering kan derfor ha en innvirkning på amyloid avleiringer i AD.
Målet med studiet i artikkel 4 var derfor å undersøke effekten av GnRHa på dannelse av amyloid plakk, GnRH and GnRH reseptor (GnRHR) mRNA ekspresjon i en dobbel transgenisk
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musemodell som er predisponert for AD grunnet Arctic and Swedish amyloid beta (A4) precursor protein mutasjoner (tg-ArcSwe). Studiet viste at GnRHa behandlingen i de transgene musene tydelig affiserte genekspresjonen både på hormon – og reseptor nivå, mens effekten av GnRHa på dannelsen av amyloid plakk forble uklar.
Funnene i avhandlingen bekrefter behovet for videre studier for å undersøke nevrobiologiske effekter av GnRH og de potensielle nevrobiologiske bivirkningene av GnRHa behandling på dyr og mennesker.
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Abbreviations
AD Alzheimer's disease
ADD3 Adducin 3 (gamma)
APOE Apolipoprotein E
APP Amyloid beta (A4) precursor protein
AR Androgen receptor
ARHGAP32 GTPase activating protein 32
ATXN10 Ataxin 10
Aβ Amyloid beta
BDNF Brain derived neurotrophic factor
BSA Bovine serum albumin
CCNE1 Cyclin E1
cDNA Complementary deoxyribonucleic acid
CPP Central precocious puberty
cRNA Complementary Ribonucleic Acid
Cy3 Cyanine 3
Cy5 Cyanine 5
CYP19 Aromatase
DAVID Database for Annotation, Visualization and Integrated Discovery
ESR1 Estrogen receptor alpha
ESR2 Estrogen receptor beta
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FAST FMRIB's Automated Segmentation Tool
FLIRT FMRIB's Linear Image Registration Tool
FMRIB Functional MRI of the Brain,
FSH Follicle Stimulating Hormone
FSL FMRIB Software Library
GABA Gamma-aminobutyric acid
GABARAP GABA(A) receptor-associated protein
GABRA4 Gamma-aminobutyric acid (GABA) A receptor, alpha 4
GH Growth hormone
GLMs General Linear Models
GM Gray matter
GnRH Gonadotropin-releasing hormone
GnRHa Gonadotropin-releasing hormone agonist
GnRHI Gonadotropin-releasing hormone1
GNRHII Gonadotropin-releasing hormone2
GnRHR Gonadotropin-releasing hormone receptor
Gria1 Glutamate receptor AMPA1
Grin1 Glutamate (NMDA) receptor, ionotropic,
HPG Hypothalamus-pituitary-gonadal axis
ITGA5 Integrin, alpha 5 (fibronectin receptor, alpha polypeptide)
LH Luteinizing hormone
LHRH Luteinizing hormone releasing hormone
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LHX5 LIM homeobox 5
MAP4 Microtubule-associated protein 4
MRI Magnetic resonance image
mRNA Messenger ribonucleic acid
NCAM1 Neural cell adhesion molecule 1
NGS Normal goat serum
PBS Phosphate-buffered saline
PFA Paraformaldehyde
qRT-PCR Quantitative reverse transcription polymerase chain reaction
RIN RNA integrity number
ROIs Regions of interest
SD Standard deviation
SNTA1 Syntrophin, alpha 1
spn Spinophilin
Syn1 Synapsin1
TBV Total brain volume
TTR Ovis aries transthyretin
VGF VGF nerve growth factor inducible
WM White matter
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List of papers
Paper I- Behavioural Brain Research 242(0), 9-16. 4-1-2013.
Peri-pubertal gonadotropin-releasing hormone analog treatment affects hippocampus gene expression without changing spatial orientation in young sheep.
Nuruddin Syed, Wojniusz Slawomir, Ropstad Erik, Krogenæs Anette, Evans Neil P., Robinson Jane E., Solbakk Anne Kristin, Amiry-Moghaddam Mahmood, and Haraldsen Ira Ronit Hebold Haraldsen
Paper II: Psychoneuroendocrinology 38, 1994-2002.2013
Effects of peripubertal gonadotropin-releasing hormone agonist treatment on brain development in sheep - a magnetic resonance imaging study.
Syed Nuruddin, Muriel Bruchhage, Erik Ropstad, Anette Krogenæs, Neil P. Evans, Jane E.
Robinson, Tor Endestad , Lars T. Westlye, Cindee Madison and Ira Ronit Hebold Haraldsen.
Paper III: Psychoneuroendocrinology 38(12), 3115-3127. 2013
Peri-pubertal gonadotropin-releasing hormone agonist treatment affects sex biased gene expression of amygdala in sheep.
Syed Nuruddin, Brynildsrud Ola Brønstad, Anette Krogenæs, Steven Verhaegen, Neil P. Evans, Jane E. Robinson, Ira Ronit Hebold Haraldsen, Erik Ropstad.
Paper IV: (submitted)
Down-regulation of elevated gonadotropin-releasing hormone gene expression in hippocampus in tg-ArcSwe mice following receptor agonist treatment.
Syed Nuruddin, Gry Helen Enger Syverstad, Sveinung Lillehaug, Anette Krogenæs, Erik Ropstad, Trygve B. Leergard, Lars Nilsson, Ira Ronit Hebold Haraldsen, Reidun Torp
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Introduction
Brain development is a complex and precisely regulated process that occurs over an extended period of time (Kostovic- and Judaš, 2006; Rubenstein, 2011). This developmental process is characterized by sex specific changes in cognitive performance, behavior and social ability (Bethke et al., 2009; Zeng et al., 2005). These cognitive and behavioral changes are also accompanied by structural and molecular changes in the brain (Dumas, 2005; Luna et al., 2001;
Paterson et al., 2006; Shaw et al., 2006). Furthermore, these structural and molecular changes which are important for normal development, may also be associated with increased neuropsychiatric disorders, if disturbed (Anagnostou and Taylor, 2011; Chow et al., 2012). Brain development and functions such as cognitive ability, for example emotional control and spatial orientation, are not only influenced by age, but also sexually differentiated (De Bellis et al., 2001) and are seen in numerous species (Cooke et al., 1998). In humans, the temporal sequence of brain maturation and the formation of functional circuits are sexually differentiated; the subcortical (e.g. striatum) and prefrontal cortical regions develop at different times in boys and girls (Casey and Jones, 2010). These temporal and organizational differences in brain development are thought to potentially result in sex-specific behaviors (Casey and Jones, 2010).
It has been proposed that they may underlie sex-associated differences in the risk of developing some neuropsychiatric diseases (Bao and Swaab, 2010). This hypothesis is supported by the observation that the time of onset of neuropsychiatric disorders such as schizophrenia, autism spectrum disorders and Alzheimer’s disease (AD), correlates with major endocrine changes during puberty and menopause (Stevens, 2002; Tareen and Kamboj, 2012;Vadakkadath Meethal and Atwood, 2005;). Recently, different stages of development, such as the prenatal and pubertal
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periods, have received more attention with regard to their organizational impact on brain development and behavior (Berenbaum and Beltz, 2011). In particular, the pubertal period has been noted as a time during which significant neuronal changes may be correlated with large changes in reproductive hormone production and secretion patterns (Blakemore et al., 2010;
Pfaff, 2009; Sisk and Zehr, 2005). The timing and magnitude of these neuronal changes may be important in directing earlier organizational activities within the brain (Kalynn et al., 2013). To investigate underlying pubertal hormonal mechanisms that might underlie sex differences in brain development and cognitive functions, some studies have focused on the modulatory effect of the sex hormone precursor, gonadotropin-releasing hormone (GnRH) on cognition and psychomotoric activity (Bryan et al., 2010; Grigorova et al., 2006).
Gonadotropin- releasing hormone and its receptor
GnRH or GnRHI is a decapeptide,an evolutionary old, 10 amino acid neurohormone that plays an important connective role between the neuronal and endocrine system (Skinner et al., 2009;
Tsai, 2006). This first GnRHI isoform was discovered and described in the mammalian brain (Cheung and Wong, 2008). The second type of GnRH or GnRH-II was first identified in the chicken brain and is referred to as chicken GnRH-II (Millar et al., 2001). It is also highly conserved among vertebrates, including mammals (Chen et al., 1998). GnRH-II specifically plays a role as a potent inhibitor of potassium channels in the amphibian sympathetic ganglion, and inhibition of these ion channels facilitates rapid excitatory transmission of conventional neurotransmitters which might provide a general neuromodulatory mechanism for GnRH-II in the nervous system (Millar et al., 2004). In many vertebrate species, a third form of GnRH is
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present and is designated as GnRH III. GnRH-III is localized in the terminal part of the olfactory neuronal cells in the brain (Millar et al., 2001).
GnRH receptors have the characteristic features of G-protein coupled receptors (GPCRs). The amino acid sequence of the GnRH receptor was first revealed for the mouse receptor cloned from the pituitary αT3 gonadotrop cell line (Tsutsumi et al., 1992). This sequence was confirmed (Reinhart et al., 1992) and provided the basis for the cloning of GnRH receptors in the pituitary glands of the human (Chi et al., 1993) sheep (Illing et al., 1993) and pig (Weesner and Matteri, 1994) which share over 80% amino acid identity. The identification of structural variants of GnRH, the discovery of their cognate GnRH receptor types in lower vertebrates and some mammals, and the influence of ligands and the intracellular milieu of signaling pathways are providing considerable insight into novel physiological and pathophysiological roles of GnRHs in diverse processes. In human, mouse and some ungulates the GnRH Type II receptor is non- functional as a GPCR. Nevertheless, GnRH II is able to bind the Type I receptor and signal in a manner distinctly different from GnRH I. A detailed molecular delineation of the interaction of GnRH variants and GnRH analogs with GnRH receptors in different cellular environments is contributing to the development of novel GnRH therapeutics.
Functional roles of mammalian GnRH and its receptor s
Functions in the reproductive system
It is well known that GnRH is primarily synthesized and secreted by neuroendocrine cells in the preoptic area of the hypothalamus and transported along axons into the hypophyseal median eminence (Harrison et al., 2004) where it is released into the hypophysial portal vasculaturees
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GnRH binds to its target, the GnRH receptor (GnRHR) on pituitary gonadotropes cells to stimulate the synthesis and intermittent release of the gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which in turn stimulate gametogenesis and gonadal hormone synthesis (Conn et al., 1986; Kasten et al., 1996; Rama and Rao, 2001). Feedback mechanisms by sex hormones regulate hypothalamic GnRH and pituitary gonadotropin release via homeostatic mechanisms. This hormonal cascade of events constitutes the well-documented hypothalamus-pituitary-gonadal axis (HPG) and this axis plays a pivotal role in the initiation of puberty and controls reproductive and behavioral function (Daniel, et al., 2011). GnRH, and its receptor gene expression in gonadotropes are critical for GnRH signaling and hence for gonadotropin secretion and sexual development. The importance of GnRH for sexual development has been shown by a strong induction of the gene and its receptor during the infantile period, followed by a weaker persistent activation during puberty in the female rat (Plant, 2001; Zapatero-Caballero et al., 2004).
Figure 1: Classical view of the organization of the Hypothalamus pituitary gonadal axis (Source:
Catherine rivier laboratory; http://pblcr.salk.edu/research.php)
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The expression of GnRH and/or its receptor in gonads (McGuire and Bentley, 2010) may modulate various processes or mechanisms during the fertilization process. The presence of GnRH and GnRH receptors have been shown to play a role in autocrine and paracrine regulation, gonadal steroidogenesis, follicular atresia, and mediation of apoptosis during spermatogenesis, sperm maturation, and fertilization (Ramakrishnappa et al., 2005).
Function in the central nervous system
Whereas the fundamental role of pituitary GnRH receptors in reproductive function is undisputed, their presence in peripheral and central tissues, unrelated to reproduction, is less known and their function poorly understood. GnRH receptors have been reported in the heart, kidney, liver, skin, bladder, hippocampus, amygdala, central grey, cerebellum, and other tissues in various species (Coit et al., 2009; Cheng and Leung, 2005; Dong et al., 2011; Harrison et al., 2004; López de Maturana et al., 2007; Skinner et al., 2009; Xing et al., 2009;). Thus, the expression of both GnRH and its receptor in multiple mammalian non-pituitary tissues and cells suggest numerous and diverse autocrine, paracrine and endocrine extra-pituitary functions. These include neuronal migration during development (Romanelli et al., 2004), neuromodulation in the brain to affect sexual behavior (Millar, 2005), neuronal plasticity in the brain (Schang et al., 2011) and inhibition of gastric acid secretion (Chen et al., 2005), cardiac development and function (Skinner et al., 2009).
Over the last three decades, it has been also shown that GnRH can affect the central nervous system. GnRH has been shown to affect hippocampal (Albertson et al., 2008; Farn et al., 1999;
Jennes et al., 1988; Osada and Kimura, 1995; Yang et al., 1999), amygdalar (Albertson et al. , 2008; Jennes et al., 1988), cerebellar (Renaud et al., 1975), preoptic (Pan et al., 1988), and
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cortical (Renaud et al., 1975; Xu et al., 2004) neurons in mice, rats and sheep.GnRH projections are wide spread in the brain (Buma, 1989; Richardson et al., 2004); Furthermore, GnRH /GnRHR expression in several areas within the central nervous system provide indication of its more extended role in brain function.
Among the different regions of the central nervous system, the hippocampus consistently expresses high levels of GnRH receptors (Skinner et al., 2009). GnRH receptor-immunoreactive neurons were found within the pyramidal cell layer, dentate gyrus and indusium griseum of the human, mouse and sheep brain (Albertson et al., 2008;He et al., 1999; Kubek et al., 1979; Lu et al., 1999; Osada and Kimura, 1995; Wilson et al., 2006). This region is the most important integrative central nervous area for cognitive, endocrinological and behavioral processes. The long lasting enhancement of synaptic transmission can be induced by activation of GnRH receptors that is mediated by ionotropic glutamate receptors in CA1 pyramidal neurons of rat hippocampal slices. Furthermore, GnRH potentiates the excitability of hippocampal and cortical neurons which are crucially involved in learning and memory (Wang et al., 2010). It has been also reported that hippocampal GnRH receptor-expressing neurons co-express estrogen receptor beta (ERβ) in sheep (Albertson et al., 2008). Interestingly, it has been reported that GnRH is likely to be elevated during the post-menopausal period in such regions (Gore et al., 2004).
Therefore, GnRH action on these neurons may contribute to the neurodegenerative pathology that accompanies Alzheimer’s disease.
In addition to the presence of extensive GnRHR neurons in hippocampal regions, these neurons are also evident throughout the olfactory system in the rodent (Albertson et al., 2008; Choi et al., 1994; Jennes et al., 1997). These structures include amygdala, mitral cell layers of the olfactory,
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accessory olfactory bulbs, piriform cortexand tenia tecta. Among these structures, the amygdala is the region where GnRH receptor expressing neurons are most widely distributed (Albertson et al., 2008). Nevertheless, there are some controversial reports about the distribution of GnRH binding sites in the rat amygdala. Some authors have reported a limited distribution, while others have detected a high density of potential GnRH receptor expressing neurons in the mouse and rat amygdala (Granger et al., 2004; Haour et al., 1987). It is postulated, that GnRH may access these receptors through neurons that project directly to the amygdala (Sanchez and Dominguez, 1995).
An increase in plasma gonadotropins after electrical stimulation of the amygdala in rats and cats has been reported, which is attributed to a direct connection between the amygdala and the preoptic area (Layton et al., 1981; Sirett et al., 1986; Velasco and Taleisnik, 1969;). Furthermore, a dense population of GnRHR-immunoreactive neurons was detected throughout the amygdala (Albertson et al., 2008) of adult mice, as with the hippocampus, elevated levels of GnRH in the amygdala provides further support for multifunctional physiological or pathological roles of GnRH. In addition to the olfactory system, GnRH receptor expression in the superior colliculus, red nucleus and cerebellum may also suggest that GnRH modulates motor control (Albertson et al., 2008)
Functional roles of hippocampus and amygdala in cognition
Hippocampus
The hippocampus and amygdala are key structural elements of the limbic system and have been investigated as mediators for learning, memory and emotional regulation and control (Ortu et al., 2013; Phillips et al., 2003). The hippocampal regions (the CA fields, dentate gyrus, and subicular
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complex) are parts of a system of anatomically related structures in the medial temporal lobe that is important for generation of new neurons (Kheirbek and Hen, 2011) and contribute to diverse memory formation. The rate of hippocampal neurogenesis is positively correlated to hippocampal-mediated learning abilities (Drapeau et al., 2003) and Hippocampal synaptic plasticity is believed to be the mechanism underlying certain types of learning and memory (Bliss and Collingridge, 1993). In this regard, neuroimaging studies have provided evidence that the hippocampus becomes more active during spatial navigation in humans, and hippocampal morphology can be affected by learning of spatial navigation skills (Mark, 2002). Furthermore, studies have shown that damage to the hippocampus impairs the acquisition and retrieval of information required to navigate in spatial mazes in rats (Jarrard, 1978; Morris et al., 1982). The left hippocampus, in particular, appears to be a key component in the retrieval of spatial memory (Spreng and Mar, 2012), and though actions with other areas of the brain makes memory recall possible.
Amygdala
Extensive empirical evidence suggests that the amygdala is a key region of the brain involved in underpinning emotions (Schaefer and Gray, 2007). Animal models investigation of amygdala function have emphasized its role in emotional learning and demonstrated that the amygdala is critical for the acquisition, storage, and expression of conditioned fear responses (Cahill et al., 1995; Cahill et al., 1999; Maren, 2001). Studies on the cognitive neuroscience of emotion and memory have demonstrated a range of means by which emotion can change the formation and recollection of episodic memory. It has been suggested that emotion, through the amygdala’s influence, can alter three components of episodic memory: encoding, consolidation and the
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subjective sense of remembering. Although episodic memory critically depends on other brain regions, most notably the hippocampal complex (Eichenbaum, 2002), the amygdala may be important for modulating the neural circuitry. Despite the fact that the amygdala and hippocampus have independent memory systems, their interaction seems to be crucial because an emotional stimulus increases the memorial impact (Phelps, 2004).
Figure 2: Magnetic resonance images of sheep amygdala and hippocampus in axial view (Source: Nuruddin et al., 2013). Bilateral red colored areas indicate the amygdala and the yellow colored areas indicate the hippocampus.
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Role of GnRH and its receptor in cognitive function
In recent years, research has focused on the effects of gonadotropin-releasing hormone (GnRH) and its receptor on sex-specific cognitive and physiological patterns, initiated by the fact that GnRH receptor expression has been found in various brain areas and peripheral tissues unrelated to reproduction (Skinner et al., 2009; Wilson et al., 2006). Results in adult human males and females, as well as in rodents, indicate that blockage of GnRH function using chronic GnRH agonist treatment may lead to significant changes in several cognitive functions. Significant impairments in domains of visuospatial and higher-order executive control functions (Nelson et al., 2008), and an episodic increase of depressive symptoms (Schmidt et al., 2004), have been described in men. In females, GnRH agonist treatment is associated with a decline in working memory (Grigorova et al., 2006; Palomba et al., 2004) and disrupted encoding of episodic verbal memory (Craig et al., 2007). Furthermore, animal models of Alzheimer's disease show that blockade of GnRH signaling by GnRH agonist had positive effects on cognitive function (Bryan et al., 2010). However, these studies have only reported effects in adults. Comparative studies in children and adolescents are lacking (Carel et al., 2009) and it has been suggested that this is due to our insufficient understanding of the biological mechanisms of sex-specific brain development during puberty (Jazin and Cahill, 2010). Recent research has suggested that GnRH plays a vital role in controlling the extent of the specificity of brain function, and changes in the availability of this decapeptide during critical periods of brain development (such as at puberty) may be reflected in altered sex-specific behavioral and physiological patterns (Wojniusz et al., 2011) in later life. Nevertheless, the exact neurobiological mechanism by which GnRH might affect brain development during puberty is not fully understood.
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GnRH agonist
Pharmaceutical GnRH agonists (GnRHa) are synthetic peptides that bind to and activate the endogenous GnRHR. GnRH agonists have been developed to be more potent than natural GnRH peptide due to the fact that they have a higher affinity to GnRH receptors and a longer half-life (Conn and Crowley, 1994). The natural GnRH has a shorter half-life because of the rapid cleavage of the bonds between amino acids at positions 5-6, 6-7 and 9-10. Synthetic GnRH agonists are derived from native GnRH by substitution of a D-amino acid for the native L-amino acid position 6 in the decapeptide. This substitution at position 6 helps the agonist resist degradation and increases its half-life and the time of receptor occupancy. Under normal conditions pulsatile secretion of GnRH from the hypothalamus is required to stimulate the release of FSH and LH from the pituitary gland as, upon stimulation, receptors are internalized and the interval between pulses allows receptor concentration to be replenished. A continuous administration (subcutaneous/intramuscular/intranasal) of GnRH agonists (goserelin, leuprolide, nafarelin, buserelin, triptorelin) initially induce a sharp increase in pituitary secretion and serum level of FSH and LH (the flare effect), which stimulates an increase in serum sex steroids (within 3 days of initial treatment). However, continuous stimulation of the pituitary by chronic administration of a GnRH agonist produces an inhibition of the hypophyseal-gonadal axis due to a down-regulation of pituitary receptors for GnRH, and decreased levels of LH, FSH and sex steroids within 2-4 weeks (Schally et al., 2001).
Application of GnRH agonist in adults and their side effects
In adults GnRH- agonists are applied in the treatment of various sex hormone dependent conditions including endometriosis (Rothman and Wierman, 2007), prostate cancer (Tammela,
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2004), breast cancer (Mastro et al., 2011) and suppression of the LH surge for use in in-vitro fertilization protocols (Zafeiriou et al., 2000). GnRHa has also been implemented in the treatment of AD patients (Bowen et al., 2004; Wang et al., 2010). The major side effects of GnRHa therapy have been primarily explained as a result of withdrawal of sex hormones. Men with prostatic cancer, receiving GnRHa therapy report physical discomfort, (Potosky et al., 2001) and lowered quality of life (Alibhai et al., 2006), reduced bone mineral density (Diamond et al., 2004), increased risk of cardiovascular events including myocardial infarction and cardiovascular mortality (Bourke et al., 2012; Levine et al., 2010) and decreased cognitive functions (Nelson et al., 2008).
GnRHa treatment in women with breast cancer is also associated with a variety of side effects including hot flushes, decreased libido, mood swings, increased cardiovascular risk and skeletal- related conditions such as bone loss (Tan and Wolff, 2007). A decline in working memory has also been reported in women treated for benign gynecological problems (Grigorova et al., 2006).
Usage of GnRHa in pediatric medicine
In pediatric medicine, GnRHa is mainly used in the treatment of central precocious puberty (CPP) and developmental disorders of the genital system. Puberty is defined as the biological, social and cognitive maturation to adulthood where the individual becomes capable of sexual reproduction to enable fertilization (Marcell, 2007). The pubertal transition is initiated and driven by increased GnRH secretion into the hypophysial portal circulation leading to downstream activation of the HPG axis (Partsch et al., 2002). Precocious puberty (CPP) is defined as the development of pubertal changes, at a youngerage than the accepted lower limits for the age of onset of puberty. Specifically, CPP is characterized by early onset of secondary sexual
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characteristics in girls who are less than eight years old and in boys who are less than nine years old (Cesario and Hughes, 2007). CPP is associated with mental and behavioral problems ranging from depression, anxiety and eating disorders to increased risk- taking behaviors as well as reduced adult height (AH) due to earlier epiphyseal closure initiated by rising sex hormones (Baker et al., 2012; Carel et al., 2004; Graber et al., 2004; Negriff et al., 2011; Stattin et al., 2011) Nevertheless, whether comorbidity is correlated with the treatment or the condition itself has not been properly investigated. To a lesser degree GnRHa treatment is used in several other diseases including idiopathic short stature, severe hypothyroidism, growth hormone deficiency or congenital adrenal hyperplasia (Carel et al., 2009).
GnRHa treatment in children is generally considered to be safe and well tolerated, No clear evidence of predicted side-effects, such as increased body mass index and reduced bone mineral density, headaches and hot flashes, have been documented (Carel et al., 2009). However, little is known about the ability of GnRHa treatment to prevent or affect development of mental and behavioral problems in children. A tendency towards fewer difficulties in coping with precocious puberty in treated CPP patients vs. untreated has been previously reported (Xhrouet-Heinrichs et al., 1997); however this study was hampered by a very small control group consisting of five untreated CPP patients that were compared to 15 GnRH analog treated CPP girls.
In neuropsychiatric research to investigate GnRH treatment effects in children, one of the main challenges is that study design is limited in such studies. To explore the effects of treatment on behavior, cognitive function and emotion regulation, an ideal design would be a randomized controlled trial (RCT). However, since GnRH agonist treatment in CPP is well established and considered to be somatically advantageous, it is unethical to randomize CPP patients into a non-
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treatment group for comparison reasons. In practice, this limits the design possibilities to longitudinal, cross-sectional or case-control studies. A longitudinal design where CPP children are assessed several times; before, under, and after cessation of the treatment, and compared to other types of patients or healthy children, assessed at the same time points, is an acceptable alternative. Nevertheless it is still difficult to interpret whether the observed effects in such studies are a result of the progressing condition or the GnRHa therapy. To address this limitation, it is necessary to use an animal model. In contrast to human models, animal studies provide the opportunity for systematic experimental manipulation of genetic and environmental variables in a RCT setting. Although the applied outcome measures and their neural substrates are not always directly applicable in humans, there are considerable similarities between pubertal and adolescent developmental processes in mammals and humans considering behavioral, neural and hormonal characteristics (Spear, 2004).
Aim of the thesis
Manipulation of GnRH receptors for clinical purposes has been used in adult and pediatric medicine for several decades with the goal of blocking sex hormone production. Existing research has concentrated on the specific effects of GnRHa treatment on the targeted disease or its influence on reproduction, with little focus on potential side effects of this treatment regime on cognitive function, emotion regulation, structural brain organization or cellular and molecular changes during brain development (Carel et al., 2009). These potential side effects of GnRHa treatment are of significant clinical importance, as many neuropsychiatric disorders, behavior and emotional problems are first observed during the peri-pubertal period. The principal aim of this thesis was to investigate effects of peri-pubertal GnRHa treatment on brain development to
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shed light both on the potential roles of GnRH and GnRHR in structural, neurobiological and cognitive functions, in brain regions that are unrelated to reproductive function.
Secondary aims were to investigate the effects of GnRHa treatment on
Endocrine and synaptic plasticity related gene expression levels in the hippocampus and any association with spatial orientation behavior.
Structural volume of total brain and other regions of interest such as the hippocampus, amygdala, white matter and grey matter.
Molecular mechanism and gene expression levels in amygdala that might underlie treatment induced structural changes in this structure.
Expression levels of GnRH and its receptor GnRHR in the hippocampus as well as beta amyloid (Aβ) deposition in the thalamus, cerebral cortex and hippocampus, in an AD mouse model.
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Materials and Methods
Animals and treatment
For paper (I, II, III), the study was conducted at the University of Glasgow’s Cochno Research Centre (55° 55’N) and all animals procedures were approved by the University’s Welfare and Ethics Committee, and in accordance with Home Office regulations (PPL 60/3826). To eliminate the possible developmental effects of steroid transfer between siblings of different sexes and reduce genetic variation, the whole study was conducted using 46 pairs of same-sex twin lambs (Scottish Mule Texel Cross,22 female and 24 male). Lambs were born between 17th March and 1st April 2008, and remained with their dams until weaned at about 12 weeks of age. Males and females were maintained separately during the entire study period. Within each set of twins, one was randomly assigned, at birth, to the control (C) and the other to the treatment (T) group.
Animals in the treatment group received subcutaneous implants of the GnRH agonist, goserelin acetate (Zoladex®; kindly donated by Astra Zeneca; Macclesfield, UK 3.6 mg) every 4 weeks from 8 weeks of age in males and 28 weeks of age in females because of the sex-specific timing of puberty in this species (Wood and Foster, 1998). The Zoladex® implant was administered in the axillar region, using a ‘SafeSystem’ needle and syringe. Animals were maintained in accordance with normal husbandry conditions, i.e., on grass, at all times except for periods of behavioral testing at approximately 8, 28 and 48 weeks of age, when they were group housed indoors. Every four weeks, throughout the study, animals were gathered and held, for no more than two hours (with their mothers, prior to weaning), in a specialized sheep handling facility at the University of Glasgow’s Cochno Research Center where blood samples were collected from
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the jugular vein, physical measurements taken (weight, shoulder height, girth) and agonist administered (where required). To guarantee that GnRHa treatment blocked pubertal development, plasma samples were assayed regularly for testosterone (males) and progesterone (females), monthly measurements of scrotal volume made during the animals’ life and the testes or ovaries excised, weighed and histologically evaluated after euthanasia at 12 months of age (March 2009) (Robinson et al., in press)
For paper IV, we used transgenic mice (Tg-ArcSwe) carrying a human AβPP cDNA with the Arctic (E693G) and Swedish (KM670/671NL) mutations and wild-type mice. Animals (Tg- ArcSwe and wild–type mice) were injected subcutaneously with 25 ng/g of the GnRH-agonist Leuprorelin acetate (Procren Depot “AbbVie”) dissolved in physiological saline, or vehicle alone. The injections were given once every fourth week from the age of 4 months, before plaque deposition has begun (Lord et al., 2006).Of the animals included in the study, about 20 % died of unknown causes before reaching the age of 12 months. This included both treated and untreated animals, leaving us with the number of animals referred to in Table 1. The remaining animals were anaesthetized using Isofluran Baxter (Isoflo™, Abbot Laboratories Ltd) and sacrificed by decapitation at 12 months, after 8 months of treatment. In addition to this, 4 month- old animals, who did not receive any pharmacological intervention, were sacrificed. Details are shown in Table 1. The tissues from all sacrificed animals were stored at -80 ºC for further use.
33 Table 1. Animals involved in the paper IV study.
Age (in months) 12 12 4
Treatment
(Leuprorelin acetate; 25ng/g) Treated Untreated Untreated
Tg - ArcSwe males 3 6 6
Wild-type males --- 6 6
Tg - ArcSwe females 7 6 6
Wild-type females --- 6 6
--- indicate tissue not analyzed
Methods
The sheep used in the studies of articles I-III were subjected to several behavior experiments at three different time points to assess their cognitive, emotional and behavioral development.The results from all of these behavioral tests are not included in this thesis. In a subsample of 30 animals (16 males and 14 females, half of them treated), spatial orientation, the expression of 17 genes within the hippocampus and the transcription profile within the amygdala (by microarray) were analyzed to ascertain if they were affected by GnRHa treatment or sex. The brains of 41 sheep (17 treated; 10 females and 7 males, and 24 controls; 11 females and 13 males) were used for morphometric analysis of different regions of the brain by using Magnetic resonance image (MRI). The mRNA expression of GnRH and GnRHR (study-IV) were analyzed by quantitative real time PCR (qRT-PCR) by using hippocampal samples from the Tg-ArcSwe mouse model and its respective controls. Furthermore hippocampus, cerebral cortex and thalamus from these animals were used for imunohistochemical studies of amyloid beta deposition.
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Behaviour study - Spatial orientation task
Spatial orientation is an essential cognitive function because most mammals depend on it for finding food, mates, and avoiding becoming prey (Wolbers and Hegarty, 2010). Animal and human studies have demonstrated that robust sex-differences exist in spatial abilities, usually favoring males (Jonasson, 2005; Kerns and Berenbaum, 1991; Terry, 2009). The main goal of this experiment (Paper I) was to explore the sexually differentiated nature of spatial orientation ability and to establish whether pre-pubertal GnRHa treatment interfered with the development of this function.
Spatial orientation was assessed at 48 weeks of age (when males and females had received GnRHa treatment for 40 and 20 weeks respectively) by means of a spatial maze task. The spatial maze setup (Figure 3) was based on the design developed and validated for use in sheep by Lee and colleagues (Lee et al., 2006)
The dividing walls of the maze were made of metal penning that was familiar to the animals and through which the test animals could see the audience pen. The outer walls of the maze arena were solid. For testing, animals were separated into smaller groups and sequentially placed in the audience pen. Individual ‘test’ animals were removed from the audience pen by a trained and familiar handler and calmly led to the entrance of the maze.
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Figure 3: Diagram of the spatial maze used at 48 weeks of age (Paper: I).
Each sheep was given 300s to traverse the maze and the test was deemed completed when the animal passed line C (Figure 3). The time taken to reach lines A, B and C were recorded as was the vocalization rate (VR) in each of the three sections of the maze, calculated as a number of vocalizations per min. If an animal did not complete the maze within 300s, it was moved back through the maze and exited through the entrance gate. Animals were tested in the same sequence over three successive trials. Trial one and two were carried out on the same day and trial three was performed on the next day.
Hippocampal gene expression involved in synaptic plasticity and endocrine signaling
In paper I of this thesis, hippocampal gene expression analyses were performed using tissue collected from animals maintained in the model outlined above and the results discussed in the context of spatial orientation, a hippocampus dependent cognitive function (Stella et al., 2012).
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Specifically, animals were sacrificed at approximately 50 weeks of age, with an overdose of barbiturate (Somulose 1ml/kg body weight; Decra Veterinary Products, Shrewsbury, UK), decapitated and their brains removed. The hippocampus was then carefully dissected from the right and left hemispheres, and the right and left hippocampi individually trisected, perpendicular to the long axis of the hippocampus, allowing isolation of hippocampal subregions (each containing CA1 to CA3). Samples were immediately frozen in liquid nitrogen and stored at - 75oC. Total RNA was isolated from the sub region containing CA1 to CA3, from the anterior section of the hippocampus of each animal, using TRIzol Reagent (InvitrogenTM, Paisley, UK) and processed according to standardized procedures. Samples were analyzed using quantitative real time-PCR (qRT-PCR) in a subset of 30 animals (Texel Cross, 14 female and 16 male). The expression of genes involved in synaptic transmission (Grin1, Gria1, GABRA4, Syn1, spn, BDNF, VGF), proliferation and differentiation (LHX5), structuring (NCAM1), that underlie synaptic plasticity and genes associated with endocrine signaling (GnRH I, GnRH II, CYP19, AR, GH, ESR1 and ESR2) were analyzed.
Postmortem magnetic resonance image (MRI)) for morphometric analysis of global and regional brain volumes
MRI provides many benefits relative to the assessment of the volume of specific brain areas, as it bypasses dissection and photography methods, which carry higher risks of errors in quantitative measurements (Pfefferbaum et al., 2004). In paper II of this thesis, the goal of the experiment was to explore GnRHa treatment effects on global and regional brain volumes. This experiment, was conducted using 41 brains (17 treated; 10 females and 7 males, and 24 controls; 11 females and 13 males) that were perfusion fixed with 4 % paraformaldehyde in 0.5% BSA immediately
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following euthanasia, post fixed in 4% PFA overnight and stored in 30% sucrose until examination.In order to minimize movements during scanning, each fixed postmortem specimen was suspended in agar gel, which was placed in a rectangular plastic container. Image acquisition was performed with a 3-T MRI scanner. For MRI preprocessing, the raw dicom files were converted to nifti and inspected for artifacts. Bias correction of the images was done using Freesurfer mri_nu_correct.mni to correct for intensity non-uniformity. Spurious noise outside the brain was removed, and Brain Extraction Tool (BET) (Smith, 2002) part of FMRIB’s Software Library (FSL)(Smith et al., 2004) was used to strip residual gel artifacts outside the brain.A set of anatomical regions of interest (ROIs) were manually drawn on the best quality brain MRI, of the chosen sheep, to act as a template. The manual segmentation of the amygdala and the hippocampus was done using FSLView, part of FMRIB’s Software Library (FSL) (Smith et al., 2004) by a single rater (Muriel Bruchage) blinded to any subject characteristics. The hippocampal ROI included the cornu ammonis, dentate gyrus and the subiculum, as each of these components show different histological characteristics and topographically well-ordered afferents and efferents (Nolte, 1993). A combination of T1- and T2-weighted data was used to distinguish the anterior boundaries that separate the amygdala from the hippocampus. However, precise delineation of the boundaries is difficult even at a histological level (Bergin, 1994).
Therefore, inter-rater reliabilities for each ROI were established by having two raters (Muriel Bruchhage and Syed Nuruddin) independently segment five datasets. Percent inter-rater voxel overlap was 94 % for hippocampus and 97 % for amygdala, indicating high reliability. For tissue segmentation,FMRIB's Automated Segmentation Tool (FAST) (Zhang et al., 2001) was used to automatically segment the T2-weighted MRI volumes of the individual brains into gray matter (GM) and white matter (WM). The underlying method of FAST is based on a hidden Markov
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random field model and an associated Expectation-Maximization algorithm. All segmented data were visually inspected for accuracy. Total GM and WM volumes were calculated by multiplying the number of voxels in each of the segmented classes by its voxel resolution.Four datasets (3 untreated males and 1 treated male) were excluded during the automatic tissue segmentation procedure due to imaging artifacts probably related to the formalin fixation of the brain where water mobility can be affected (Tovi and Ericsson, 1992).
Transcription profiling through microarray in amygdala samples
Based on the results from the MRI experiment which suggested changes in amygdala volume, microarray gene expression studies were conducted to explore GnRHa effects in the amygdala at a molecular level. The genome information availability and the parallel development of microarray technology have provided the means to perform global analyses of the expression of thousands of genes in a single assay (Eisen and Brown, 1999; King and Sinha, 2001). The results provide an assessment of the expression levels of the genes included on the microarray in a particular cell, tissue or organ. In Paper III of this thesis, we describe the gene expression profiles of the left and right amygdala using 8 X 15 K Agilent ovine microarrays in 30 same-sex twin lambs (14 female and 16 male), half of which were treated with the GnRHa. Two-color microarray experiments were conducted to identify genes being significantly differentially expressed due to long term peri-pubertal GnRHa treatment. The microarray experiment was conducted as a common reference design, using a reference where target samples were hybridized with a reference sample consisting of equal amounts of total-RNA from all samples.
This method reduces human error as each sample is handled the same way and compared against a reference sample which contains many samples pooled (Kendziorski et al., 2005; König et al.,
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2004). Total RNA extraction was performed according to the TRIzol manufacturer recommendations (InvitrogenTM, Paisley, United Kingdom), followed by a DNase I treatment (RNase-Free DNase Set, Qiagen, Crawley, UK) for 20 minutes at 25°C. Further RNA purification was conducted using an RNeasy Mini-Kit (Qiagen, Crawley, UK) according to the manufacturer’s recommendations. A dye swap design was applied, whereby within each group DNAse treated RNA was randomly labeled with cyanine 3 (Cy3) CTP and cyanine 5 (Cy5) CTP using a Quick Amp Labeling Kit, two-color (5190-0444, New Castle, DE) according to the manufacturers methodology. Following hybridization, washing and drying, the slides were scanned in GenePix® 4000B two-color scanner (Axon instrument, Foster City, CA). Two channel images were imported into Agilent’s feature extraction 9.1 software for features (spot) extraction and alignment.
After analysis microarray data have been submitted to the NCBI's Gene Expression Omnibus (GEO) and are accessible through GEO Series accession number GSE44202. The differential expressions of some of the selected genes from the microarray experiment were confirmed by qRT-PCR. In order to understand the biological significance of microarray results, Gene Ontology (GO) term enrichment analysis of the list of differentially expressed genes was conducted in The Database for Annotation, Visualization and Integrated Discovery (DAVID) 6.7 (Huang et al., 2008). To identify gene orthologs or homologs to sheep genes, we used a translation table provided by Agilent Technologies and converted probe IDs into official gene symbols using an in-house python script. Furthermore, to interpret the biological function of the gene lists, the Ingenuity Pathway Analysis (IPA) version 7.5 (http://www.Ingenuity.com) software packages were used for networking analyses. This software not only generates a
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biological network from a list of selected genes, but also provides biological functions and canonical pathways from HUGO ortholog names of the genes that were imported into the program. A network of genes is created when a gene regulates the function of other genes. To develop a network, the IPA software adds other genes and/or different molecules that are linked with two focus genes.
mRNA Expression of GnRHI and GnRHR in the hippocampus of Transgenic mice In paper IV of thesis, exploration of GnRHa (Leuprorelin acetate) treatment effect on murine gene expression level of GnRHI and its receptor in hippocampal samples were performed by using standard qRT-PCR methods from the sample of left hemisphere hippocampus.
Measurement of amyloid-β deposition in cerebral cortex, thalamus and hippocampus using immunohistochemistry
The right hemisphere of hippocampus, cerebral cortex and thalamus were used for immunohistochemistry study. Sagittal cryosections (25 µm; Leica CM3050 S) of tissues were performed. All sections were post–fixed with 4% formaldehyde (PFA) for 5 minutes, pretreated with 80 % formic acid for 2 minutes and 2% H2O2 for 7 minutes. After washing with 10 mM PBS, pre - incubation solution (10% normal goat serum (NGS), 1% bovine serum albumin (BSA), 0.5% Triton X-100 in 10 mM PBS) was applied to the sections for 30 minutes at room temperature. Afterwards, the sections were incubated with an Aβx-40-specific polyclonal primary antibody (0.5 μg/ml; Agrisera, Umeå, Sweden) diluted 1:2000 in primary antibody solution (3% NGS, 1% BSA, 0.5% Triton X-100 in 10 mM PBS) at 4˚C overnight. The antisera was generated and evaluated for specificity as described (Näslund et al., 2000). After washing
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steps, the sections were incubated for 1 hour with a biotinylated goat-anti-rabbit antibody (BA- 1000, Vector Laboratories, CA, USA) diluted at 1:300 in 3% NGS, 1% BSA, 0.5% Triton X-100 in 10 mM PBS, washed in 10 mM PBS, and afterwards, incubated 1 hour at room temperature with streptavidin-biotinylated horseradish peroxidase complex diluted at 1:100 in 0.5% Triton X- 100 in 10 mM PBS. After washing with 10 mM PBS, all sections were incubated with 3,3’- diaminobenzidine tetrahyprochloride (Sigma, MO, USA) for 5 minutes, before 0.1% H2O2 was added to 10 ml DAB solution and applied on the sections until proper labeling was achieved (3 minutes). The sections were briefly rinsed in water, mounted and stored at room temperature.
Then, image acquisition was done by using an automated slide scanner system (Mirax Scan, Carl Zeiss MicroImaging GmbH, Jena, Germany) for acquiring high-resolution TIFF images with a spatial resolution of 0,205 µm/pixel. Images were inspected by virtual microscopy using the Mirax Viewer tool. To compensate for differences in background color intensity following immunohistochemistry, all image histograms were normalized using the match-color algorithm in Adobe Photoshop CS6 with a photomicrograph of a wild-type section as reference (Sedgwick, 2008). Afterwards, quantitative image analysis was performed in three regions of interest (ROIs) using ImageJ 1.46r (http://imagej.nih.gov/ij). The outlines of the cerebral cortex and hippocampus were manually delineated in each section. The cerebral cortex was delineated, in an anterior plane, by a line connecting the rhinal fissure and anterior tip of the external capsule, dorsally by the external surface of the brain, ventrally by the external capsule, and posteriorly by the dorsal subiculum. The delineation of the hippocampus included the three cornu ammonis subfields (CA1, CA2, and CA3), the dentate gyrus, and subiculum, and was defined anterior to the fimbria, dorsally and posterior to the external capsule, and ventral to the thalamus. Images
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were binarized by selecting a threshold value in ImageJ which yielded boundary definitions best corresponding to the observed plaque boundaries. The same threshold value was used for all sections. The area of each ROI and the area of labeled objects within each ROI were calculated, and the results expressed as labeled area fraction in percent (labeled area/ ROI area x 100). For each animal the mean area fractions of three sections were used for the final analyses of group variation and standard deviation (s).
Statistical analyses
The analyses were conducted with the goal of exploring the sex-specific effect of GnRHa treatment on various outcome measures. The statistical methods were applied according to the experimental design and data characteristics.
Paper I
Spatial orientation
Times to traverse the spatial maze and vocalization rate at 48 weeks of age were analyzed in the same 30 animals as hippocampal gene expression. All data were logarithmically transformed because of skewed data distribution. Mixed between-within subject ANOVA was used to analyze the change in time and vocalization rate through the three successive trials and to assess the differences between T and C animals. Independent sample t-tests were used for assessment of sex differences. Since gene expression analysis provides only relative values no statistical associations between spatial orientation results and gene expression analyses could be performed.
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The analysis of hippocampal gene expression
The ΔCt was calculated from the difference in expression between the gene of interest and mean expression of the two reference genes (GAPDH and SDHA). In order to assess any effects of treatment the ΔΔCt was calculated as the difference between the ΔCt value of control and treated samples, within sexes and for both hemispheres. Sex differences were analyzed separately for control and treated animals; ΔΔCt was calculated as the difference between male and female ΔCt values. Relative gene expression, expressed as fold change was calculated by using 2-ΔΔCt. The log2 transformed fold change values (2-ΔΔCt) were used for statistical analysis. Differences in gene expression between C and T animals, as well as between males and females were evaluated by Wilcoxon signed rank test.
Paper II
MRI study for measurement of global and regional brain volume
The mean and standard deviation (SD) were calculated for the volume of the different regions of interest. Total brain volume, white matter and gray matter volume, as well as the volume of the left and right amygdala and hippocampus, were compared between groups (C vs T), while controlling for sex and treatment group using General Linear Models (GLMs). The sex by treatment interaction term was included in the models, allowing us to test for differential effects of treatment between male and female animals. The null hypothesis of no effects was rejected if p < 0.05, Bonferroni adjusted by a factor of 7 (corresponding to a nominal alpha of p < 0.007).
The assumption of normally distributed residuals was evaluated using a normal quintile (Q-Q) plot and this assumption of normally distributed residuals was met for all parameters. Statistical analyses were performed using SPSS version 19.0 (IBM Corp., Armonk, NY, USA).
